The invention relates to carbazolylvinyl dyes and their use in the staining of poly(amino acids), including peptides, polypeptides and proteins in solution, in gels and on solid supports.
Detection and analysis of poly(amino acids) is of great importance in a multitude of diverse activities, ranging from commercial enzyme production, forensics analysis and diagnostics to basic research in biochemistry, molecular biology, neuroscience, developmental biology or physiology. As used herein, a poly(amino acid) is any homopolymer or heteropolymer of amino acids, including peptides and proteins. Primarily, poly(amino acids) are detected and characterized using gel electrophoresis, by solution quantitation assays or by detection on solid supports, such as filter transfer membranes.
Unmodified protein or other poly(amino acid) bands in gels are generally not visible to the naked eye. Thus, for electrophoretic gels to be useful, the bands or spots must be stained, so that they can be localized and identified. Use of the novel carbazolylvinyl dyes possesses many advantages over known methods for staining poly(amino acids) on gels that are based on COOMASSIE Brilliant Blue (CBB), silver staining or zinc/imidazole reverse staining: Staining is very rapid, and is relatively insensitive to poly(amino acid) composition. Visualization of stained gels is possible without destaining, and the stained bands remain readily detectable for several days.
U.S. Pat. No. 5,616,502 (Merocyanine Dye Protein Stains) describes an extensive assortment of styryl dyes that are useful for staining proteins in gels, on blots and in solutions, where the styryl moiety is a substituted or unsubstituted aminophenyl, which is not a carbazole, that is chemically attached to a substituted or unsubstituted carbon-carbon double bond at the 4-position of the aminophenyl. Most commonly the amino group in the styryl dyes is substituted by two alkyl groups and not substituted by any aryl groups.
Styryl dyes sold under the trademark SYPRO Orange and SYPRO Red (Molecular Probes, Eugene, Oreg.) can detect proteins in SDS-polyacrylamide gels using a simple, one-step staining procedure that requires 30 to 60 minutes to complete and does not involve a destaining step. As little as 4 to 10 nanograms of protein can routinely be detected with SYPRO Orange or SYPRO Red dyes, rivaling the sensitivity of rapid silver staining techniques and surpassing the best colloidal CBB staining methods available. However, both SYPRO Orange and SYPRO Red dyes require 7% acetic acid in the staining solution, which is problematic when electroblotting, electroelution or measuring enzyme activity is indicated. If acetic acid is not included in the staining solution when using the SYPRO Orange and SYPRO Red stains, proteins may be recovered from gels, but the detection sensitivity obtained with these stains is substantially lower and significant protein-to-protein variability in staining is observed.
Unlike methods using the SYPRO Red and SYPRO Orange styryl dyes, methods using preferred carbazolyl dyes of the invention do not require the use of organic solvents for optimal staining of proteins, and the carbazolylvinyl dyes are readily soluble and stable in aqueous staining solutions. A preferred dye of the invention, Compound 9 (Example 9) is now available under the trademark SYPRO Tangerine (Molecular Probes, Eugene, Oreg.). The carbazolylvinyl dyes of the invention provide superior staining of proteins in SDS-polyacrylamide gels, particularly with respect to recovery of proteins after electrophoresis for electroblotting, zymography, electroelution, or in downstream microchemical characterization by techniques such as matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS), which techniques are important in the burgeoning field of proteomics. In addition, the carbazolylvinyl dyes exhibit a large Stokes shift between their absorption and emission maxima. Finally, the staining procedure of the present invention is rapid and simple, requires minimal labor, and allows the detection of as little as 1 ng of a poly(amino acid) per band; this sensitivity is in many cases equal to or better than that of rapid silver staining methods, with far less hazard and expense, and is at least an more than an order of magnitude better than CBB staining.
Methanol, a constituent of CBB stains, is one of the top five chemicals (on a total mass basis) appearing in the United States Environmental Protection Agency""s (EPA""s) Toxic Chemical Release Inventory [http://www.epa.gov/opptintr/tri/]. The dyes of the invention are environmentally benign protein stains that do not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply NaCl-containing solutions. Since proteins may be stained without the need for harsh fixatives, they are easily eluted from gels or utilized in zymographic assays, provided that SDS present during electrophoresis does not inactivate them. Methods described in this patent allow visualization of proteins and excision of small regions of a gel or even of individual bands, followed by their transfer to membranes by electroblotting. This permits use of much smaller amounts of transfer membrane and corresponding savings in immunodetection reagents. As with reverse staining procedures such as zinc-imidazole staining, the gentle staining conditions used with the newly described dyes are expected to improve protein recovery after electroelution and to reduce the potential for artifacts in protein modifications, such as the alkylation of lysine residues and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.